Energy transduction in respiration and photosynthesis involves the coupling of electron transfer reactions to the generation of an electrochemical gradient across a bilayer membrane. Although the sequence and kinetics of the electron transfer reactions are by and large known in these systems, much less is known about the spacial arrangement of the electron carriers and the distances between them. Ideally, the electron carriers could be used as probes in order to unravel their spacial organization. The long-term objective of this project is to use electron spin-lattice relaxation enhancement measurements to determine distances between redox active sites in proteins. The first objective is to calibrate the measurements on samples in which the distance between two paramagnetic centers is known. In this study, cytochrome c will be covalently modified with ethylenediaminetetraacetate (EDTA) and specific singly-labeled cytochromes c will be isolated. These samples will contain two paramagnetic sites at a fixed distance ranging from 15-24 A. The electron spin-lattice relaxation rate enhancement of the electron localized at the heme iron due to a magnetic interaction with the nearby metal ion complexed to a surface-bound EDTA will be systematically investigated by varying the metal ions. The second objective is to use measurements of electron spin-lattice relaxation rate enhancements to determine the spacial organization of redox sites in Photosystem II, a system for which such distance information is not known. Three types of applications will be pursued in order to unravel the spacial organization of the redox active sites in Photosystem II and also to establish the general applicability of this method to determine distance in other systems. The first application is to measure magnetic interactions between endogenous sites. The second is to measure interactions between endogenous sites and a Dy3+-inhibitor derivative bound specifically to Photosystem II. The third is to label polypeptides of Photosystem II with Dy3+, reconstitute the polypeptides, and use measurements of electron spin-lattice relaxation rate enhancements of endogenous sites to determine the spacial organization of these polypeptides. These experiments will provide a basis for using electron spin-lattice relaxation measurements to determine distances in other redox protein complexes.